Many experimental therapies promising to conquer cancer are being developed. However, for now and for the foreseeable future, the main treatment for disseminated cancer is cytotoxic chemotherapy. The biological information explosion now underway holds great promise for improving cytotoxic chemotherapy. Chemotherapeutic agents that trap topoisomerases in complexes with DNA are widely prescribed and extremely valuable for the treatment of numerous disseminated cancers. Nonetheless, they often fail to initially reduce tumors, or first successfully shrink tumors, but then fail to prevent regrowth due to acquired resistance. The reasons why these drugs fail to cure cancers remain poorly understood; there is a great need to improve their efficacy. The applicant's research is focused on the long-range goal of improving therapy with topoisomerase-targeting drugs. As a first step, there is a critical need to obtain detailed knowledge of the pathways that are exploited by tumors to evade these drugs, and learn how these pathways differ between normal and tumor cells, and among specific molecularly characterized tumor subtypes. The proposed research focuses on the molecular responses to camptothecin-DNA-topoisomerase I complexes. Previous research discovered that within minutes of treatment, camptothecin causes topo I to be specifically ubiquitinated and degraded. The role of the drug-induced, ubiquitin-dependent downregulation of topo I in relation to the anticancer action of camptothecins remains unexplored. The specific aims of the proposed research are to determine the role of the downregulation of topo in intrinsic and acquired resistance to camptothecins, and to identify the molecular basis of the ubiquitination in the downregulation of topo I. Identification of the molecular basis of the downregulation is essential for providing direct experimental evidence for its involvement in resistance. This research is expected to characterize a new mechanism for regulating topo I and the topo I-DNA complex and identify a molecular basis for resistance that involves downregulation of topo I or ubiquitin-dependent repair. Obtaining this information is a necessary first step toward predicting molecular subtypes of tumors that will respond to therapy, and identifying and developing synergistic drug targets for curing more cancers with this widely prescribed class of chemotherapeutics. The current and future ability to molecularly phenotype tumors is leading to a new generation of cancer diagnostics for identifying altered pathways in tumors. These diagnostics are expected to enable specific therapies to be applied based on a tumor's molecular or metabolic fingerprint. In order to design these therapies it is essential to characterize the pathways affecting chemotherapeutic drug responses.